Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.

In order to establish a novel serologic test—Luciferase immunoprecipitation system ( LIPS) , full?length prM?E of TBE virus was expressed by eukaryotic expression system and its effect was evaluated by LIPS technology. Firstly, using TBEV′s RNA as template, prM?E gene of TBEV was amplified by RT?PCR, eukaryotic expression vector pNLF?prM?E was constructed through inserting prM?E gene into pNLF1?secN vector. Fusion protein prM?E?luciferase was expressed by transfecting Cos7 cells. The expression of fusion protein was measured by testing values of luciferase in supernatant of Cos7 cells. Immunofluorescence assay ( IFA) was employed to identify the specificity of the recombinant protein and LIPS was used to detect the serum of TBEV infected patients. High expression level of luciferase was tested in the supernatants of the transfected cells. The specific binding of prM?E with TBEV antibody was confirmed by IFA. The LIPS assay detected 19 positive results out of 20 sera of the TBEV infected patients, 7 control sera all showed negative results. The results indicated that the expressed prM?E had good sensitivity and specitivity, which could be used as a candidate diagnostic antigen for LIPS assay for TBEV infection.